4 Results and discussion
4.2 Impact of additives on protein profile and processing behaviour
Hot compression moulding of unmodified crambe and carinata industrial oilseed meals (Figure 3, 4) indicates that there may be room to improve properties by modifying the processing environment; even the best conditions resulted in over 20% protein solubility (Paper I). In order to affect the outcome of hot compression moulding a series of additives were studied in order to assess their effect (Table 1). The additives chosen are fairly benign from an industrial health and safety point of view and can be renewably sourced. An exception to this is Jeffamine™ (JF) which may be toxic but was chosen to provide thermally stable and reactive amine sites compared to U.
Additives that raise pH (NaOH and ammonium hydroxide (AH)) were found to have the largest effect on crambe and carinata meal-based materials (Figure 7, Paper II). Solubility decreased with lower additions of NaOH (1.5, 3 parts per hundred parts of 70:30 meal:glycerol (pph)) and at all levels of AH. At higher levels of NaOH (4.5 pph) an increase in both high MW and low MW in the extractable proteins compared to the control indicated protein breakdown at our pressing conditions (130 oC, 30% glycerol). Basic conditions are known
to promote protein-protein cross linking such as isopeptide and lanthionine cross links (Rombouts et al., 2013; Rombouts et al., 2010; Friedman, 1999). Sugars that are also present in the meals (Pedroche et al., 2004; Steg et al., 1994) could form Maillard type cross links, are also promoted by a basic environment (Singh, 1991). In AH these effects do not change appreciably with dose, perhaps due to the mechanism being saturated at the initial dose and AH not being a strong enough base to result in degradation at high doses.
Table 1. Additives for modifying hot compression molded crambe and carinata oilseed meal with their proposed action.
In aqueous protein extracts from crambe the modification of pH to 10.5 (NaOH) in solution after extraction (C2, Paper IV) resulted an increase in the high MW fraction compared with the unmodified extract (C1, Paper IV). Despite this shift of MW in the concentrate with increased pH, the outcome after thermal processing was not affected by the pH modification (hot pressed C1 vs. hot pressed C2, Paper IV). This indicates that the starting composition, which is identical in C1 and C2, plays a larger part than aggregation before pressing induced in C2. This may also be due to a lower level of alkali present during pressing of C2 compared to the oilseed meals of Paper II.
Combinations of CA and NaOH were added to the oilseed meals in order to examine the possibility of CA cross linking as observed in other protein systems (Xu et al., 2015; Reddy et al., 2009). At a constant level of CA (3 pph) and varying NaOH (1.5, 3, 4.5 pph) the lowest level of NaOH addition resulted in high solubility for crambe, as in the case of CA alone (Paper II supplemental data). Higher levels of NaOH resulted in small changes in solubility and MW profile, indicating a simple pH effect (no CA cross linking) (Figure 7). A constant CA/NaOH ratio (2:1) resulted in only slight changes in solubility and
Type Additive Abbreviation Proposed Action
Base NaOH NaOH Promote protein-protein interactions
NH4OH AH Promote protein-protein interactions
Acid/Base Citric acid/NaOH CA/NaOH Cross linking
Acid Salicylic acid SA Retard protein-protein interactions
Citric acid CA Cross linking
Ascorbic acid As Improve protein behaviour
Reactive Jeffamine EDR 176™ JF Provide reactive amine sites
Benzoyl peroxide BP Thermally decomposing oxidant
Denaturant Sodium dodecyl sulphate SDS Allow protein rearrangement
Urea U Allow protein rearrangement
MW profile, with the exception of the highest level in crambe (9/4.5 pph) where the extractable high MW fraction was reduced (Figure 7). In the case of CA cross linking one would expect a dose dependant response and a marked departure from the control, which was absent in our case.
Recent work has highlighted the importance of deprotonating the CA for successful protein cross linking (Xu et al., 2015) and it is unknown if this condition has been met during pressing oilseed meals as a plasticized solid. The use of CA alone has been shown to fail in cross linking of zein protein (Selling & Sessa, 2007) and did not result in any improvement in the MW profile at any level used, showing higher protein solubility than the raw unprocessed meal in both MW fractions (Paper II supplemental information). The additives surveyed not represented in Figure 7 were found to have only a small effect on the protein behaviour (As, SDS, SA) or resulted in an increase in protein solubility (U, SB, CA) (Paper II supplemental information).
In examining the samples with reduced solubility due to the series of additives, the bulk of the decreases come from the high MW end of the MW
Figure 7. Protein solubility of hot compression moulded crambe and carinata oilseed meals
(30% glycerol, 130 oC, 10 minutes), divided into high MW and low MW fractions by SE-
HPLC. Solubility is expressed as a % of the total solubility of unpressed raw meal. Error bars denote one standard deviation. Adapted with permission from Paper II, Copyright 2014 American Chemical Society.
-60 -40 -20 0 20 40 Sol ubi lit y (% ) -60 -40 -20 0 20 40 Co nt ro l 2. 5 5 7. 5 1.5 3 4. 5 1.5 3 4. 5 0.5 1.5 3 0. 5 1 1. 5 0. 44 0. 89 1. 33 AH NaOH C(2x)/N(x) C(3)/N(x) JF BP Sol ubi lit y (%) high MW, low MW Crambe Carinata
range demonstrating a higher tendency of the high MW protein fraction to form aggregates compared to the low MW fraction (Figure 7). A similar trend was seen for purified proteins when small molecules were not removed by dialysis (section 4.1).
Previous work has shown that extrusion of crambe and carinata oilseed meals is difficult to achieve (Johansson, 2010) but processability is improved for crambe by the incorporation of WG and U (Henne, 2011). As an additive for modifying extrusion processing U levels of 15% were required for the production of acceptable films, 10% U could be extruded but not at acceptable quality for further testing (60:40 crambe:WG, 25.5% glycerol, Paper III). Under extrusion conditions U is likely to dissociate into ammonium and cyanate reacting with lysine and cystine groups of the proteins (Rombouts et al., 2013). This effectively blocks cross linking reactions with these groups in addition to contributing to denaturing the proteins (Türe et al., 2011). The effectiveness of this approach in the 60% crambe, 40% WG + 15% U system is in contrast to compression moulding of crambe meal without WG where 7.5 pph U resulted in films that were of poor quality (Paper II supplementary data).